5.2 Discussion of main results
5.2.3 Mediators of VTE risk in obesity
Papers III and IV aimed to investigate potential mediators on the casual path between obesity and VTE in order to expand our understanding of the pathophysiology of obesity-related VTE.
In paper III, we found that the adipokine leptin was associated with VTE risk, particularly in men. However, when BMI was taken into account, the apparent association disappeared. Further, when leptin was introduced in the regression models as a potential mediator for the association between obesity and VTE, the risk estimates were only marginally affected in both sexes, suggesting no mediating effect. This was somewhat surprising as previous findings based on in vitro studies speculated that leptin could play a role in obesity related-VTE. Several in vitro studies have investigated the effect of leptin on platelet activation parameters 186 and on the expression of key hemostatic factors for
coagulation and fibrinolysis.178-180,242,243 In human peripheral mononuclear cells, leptin was shown to induce TF activity, antigen and mRNA expression,178,179 as well as the release of TF-bearing microparticles. 242 Interestingly, Ayer et al. 243 demonstrated that the upregulation of TF activity in mononuclear cells was only achieved at supra-physiological concentrations of leptin, such as those used in the aforementioned studies,178,179,242 but not at the lower concentrations generally observed in obese subjects. Thus, in vitro findings may not reflect the ability of physiological levels of leptin to upregulate TF expression in vivo.
Moreover, leptin was implicated in the upregulation of PAI-1 mRNA and protein expression in human coronary endothelial cells.180 However, this is in contrast with findings in leptin-deficient ob/ob mice, which were characterized by high levels of PAI-1244 and with an in vitro study, where leptin was reported to have no effect on PAI-1 secretion from human adipocytes.245 In experimental studies, leptin was also assessed in a mouse model of venous thrombosis, in which PE was induced by the injection of collagen and epinephrine (well-known agonists of platelet activation) into the jugular vein.246 The authors found that prior administration of leptin-neutralizing antibodies was associated with improved survival and reduced number of occlusive thrombi in the pulmonary vessels. However, since thrombosis is essentially driven by platelet activation in this model, it poorly resembles the mechanism of venous thrombus formation in humans, which frequently occurs in the presence of stasis and
In previous epidemiological studies, involving a small number of participants,
circulating leptin levels have been reported to be associated with a broad range of hemostatic factors, such as platelet activation parameters, and procoagulant and fibrinolytic factors.248-251 In two larger studies (the British Regional Heart Study and the Netherlands Epidemiology of Obesity Study), leptin levels were associated with several hemostatic factors, including fibrinogen, factor VIII, factor IX, von Willebrand factor, tissue plasminogen activator and D-dimer, even after adjustment for measures of body fat.252,253 However, a main limitation of the above mentioned studies248-253 is that they are not designed to reveal the direction of the associations given their cross-sectional nature, which precludes any inference on potential causality owing to the undetermined temporal sequence between exposure (i.e., leptin) and outcome (i.e., hemostatic factors). Finally, the association between leptin and venous disease was investigated in few epidemiological studies, which suggested a potential role of leptin in DVT after knee arthroplasty, peripheral chronic venous disease, and post-thrombotic
syndrome.254-256
In light of our findings, the association of leptin with hemostatic factors reported in vitro 178-180,242,243 and in epidemiological studies 248-253 does not seem to be clinically relevant for the underlying pathophysiology of VTE in obese, and interventions aimed at targeting leptin would unlikely impact the incidence of obesity-related VTE.
In paper IV, PAI-1 was investigated as a potential mediator of VTE risk in obesity. In this paper, we found that the risk of future VTE increased in a dose-dependent manner with increasing plasma levels of PAI-1, even after adjustment for BMI and CRP, a reliable and sensitive downstream marker of inflammation. Several studies, often with limited sample sizes, have provided inconclusive results on the association between PAI-1 and VTE over the last decades.257-260 It is worth noting that the findings from two large population-based studies were also conflicting.156,261 In the Longitudinal Investigation of Thromboembolism Etiology (LITE) study, a nested case-control study comprising 308 VTE patients and 640 controls, Folsom et al. found no association between PAI-1 antigen levels and risk of future VTE,261 contrasting our findings. However, the cohorts that served as the source population for the LITE study were composed of middle-aged and old individuals at baseline,261 which could limit the comparability with our study. In the MEGA study, a case control-study involving 770 VTE patients and 743 controls, Meltzer et al. showed that high plasma levels of PAI-1 were associated with increased risk of VTE.156 The association remained, albeit attenuated,
after additional adjustment for BMI and markers of inflammation, which is in line with our results.
We found that PAI-1 mediated almost 15% of the VTE risk in obesity, which was largely independent of chronic low-grade inflammation, reflected by CRP levels. Although the mediating effect was somehow modest, PAI-1 could be a potential target for intervention to decrease the risk of VTE in obese subjects. The relevance of PAI-1 in the pathogenesis of venous thrombosis was highlighted by a murine model of DVT, in which increased
expression levels of PAI-1 were associated with larger thrombus size and impaired thrombus resolution.68 Targeting 1 in order to reduce VTE risk in obesity might be suitable as PAI-1 seems to be predominantly regulated by environmental factors.262,263 It was shown that angiotensin II, glucose, and insulin may induce the expression of PAI-1.227227 In
interventional studies, treatment with angiotensin-converting enzyme inhibitors or with oral hypoglycemic medications was associated with decreased PAI-1 levels.227,264-267 Notably, many efforts have been devoted to develop drugs that can modulate PAI-1 activity, the so-called PAI-1 inhibitors.268 Several of these inhibitors, including low molecular weight molecules, peptides, monoclonal antibodies and antibody fragments, have been extensively characterized in experimental studies.227,268 In view of the novel insights into PAI-1 biology, it may be speculated that targeting PAI-1 with inhibitors or indirectly via regulatory pathways (e.g. by the use of angiotensin-converting enzyme inhibitors or oral hypoglycemic agents) could emerge as promising therapeutic options to reduce the VTE risk in obesity.
6 Conclusions
• In the general population, almost 25% of all incident VTE cases were attributed to overweight and obesity. Given the likely causal relationship between obesity and VTE, the rising prevalence of overweight and obesity may substantially contribute to the incidence of VTE in the coming years.
• The combination of obesity and established prothrombotic genotypes, evaluated either as individual SNPs or as a GRS, had an additive effect on the risk of overall VTE.
However, such combination may have a supra-additive effect on the risk of DVT and unprovoked VTE.
• The apparent association between plasma leptin levels and VTE risk is confounded by BMI and leptin does not mediate the VTE risk in obese. These findings suggest that the effect of leptin on prothrombotic hemostatic factors reported in vitro is not clinically relevant for the mediation of VTE risk in obesity.
• Elevated PAI-1 levels, the main inhibitor of fibrinolysis, were associated with increased risk of future incident VTE and mediated almost 15% of the VTE risk in obesity in our study. Targeting PAI-1 may emerge as a promising therapeutic approach in obesity-related VTE.
7 Future perspectives
The aim of this thesis was to expand our knowledge concerning obesity-related VTE. We showed that one of four VTEs in the general population are attributable to overweight and obesity. Nevertheless, due to the increasing prevalence of overweight and obesity, we would expect this proportion to increase even further in the future, which may be evaluated in future studies to guide public health authorities.
We could not detect a synergism between obesity and established prothrombotic genotypes. Future research should aim to investigate the interaction between obesity and other genetic and environmental factors using large prospective population-based studies. Findings from these studies may facilitate the identification of overweight and obese individuals at a substantially high risk of VTE who would benefit most from public health interventions for disease prevention.
We identified PAI-1 as a mediator of VTE risk in obesity. PAI-1 might be a promising candidate to be investigated in future studies, especially because PAI-1 inhibitors have been extensively explored in experimental studies and even tested in some clinical trials, yet for conditions other than obesity and VTE.227 Nevertheless, the search for other mediators has to be pursued in the coming years as only a small proportion of the VTE risk in obesity seems to be explained by specific mediators. Investigating single plasma proteins, as we did in this thesis, may not be the most efficient way to detect further causal biomarkers or pathways in obesity-related VTE and the use of somewhat “wide-spectrum” techniques such as
proteomics, transcriptomics or metabolomics may assist in improving efficiency by which new mediators can be detected in the future.
Even though we could shed some light on obesity-related VTE mechanism, much is still unknow and obesity remains a major and challenging risk factor for VTE.
References
1. Di Nisio M, van Es N, Büller HR. Deep vein thrombosis and pulmonary embolism. Lancet.
2016;388:3060-3073
2. Line BR. Pathophysiology and diagnosis of deep venous thrombosis. Semin Nucl Med.
2001;31:90-101
3. van Langevelde K, Srámek A, Vincken PW, van Rooden JK, Rosendaal FR, Cannegieter SC.
Finding the origin of pulmonary emboli with a total-body magnetic resonance direct thrombus imaging technique. Haematologica. 2013;98:309-315
4. Sørensen HT, Horvath-Puho E, Lash TL, Christiansen CF, Pesavento R, Pedersen L, Baron JA, Prandoni P. Heart disease may be a risk factor for pulmonary embolism without peripheral deep venous thrombosis. Circulation. 2011;124:1435-1441
5. Enga KF, Rye-Holmboe I, Hald EM, Løchen ML, Mathiesen EB, Njølstad I, Wilsgaard T, Braekkan SK, Hansen JB. Atrial fibrillation and future risk of venous thromboembolism:the Tromsø study. J Thromb Haemost. 2015;13:10-16
6. Dunnick NR, Newman GE, Perlmutt LM, Braun SD. Pulmonary embolism. Curr Probl Diagn Radiol. 1988;17:197-237
7. Goldhaber SZ, Elliott CG. Acute pulmonary embolism: part I: epidemiology, pathophysiology, and diagnosis. Circulation. 2003;108:2726-2729
8. Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet. 1999;353:1167-1173 9. Wolberg AS, Rosendaal FR, Weitz JI, Jaffer IH, Agnelli G, Baglin T, Mackman N. Venous
thrombosis. Nat Rev Dis Primers. 2015;1:15006
10. Kearon C, Ageno W, Cannegieter SC, Cosmi B, Geersing GJ, Kyrle PA. Categorization of patients as having provoked or unprovoked venous thromboembolism: guidance from the SSC of ISTH. J Thromb Haemost. 2016;14:1480-1483
11. Braekkan SK, Siegerink B, Lijfering WM, Hansen JB, Cannegieter SC, Rosendaal FR. Role of obesity in the etiology of deep vein thrombosis and pulmonary embolism: current
epidemiological insights. Semin Thromb Hemost. 2013;39:533-540
12. Klovaite J, Benn M, Nordestgaard BG. Obesity as a causal risk factor for deep venous thrombosis: a Mendelian randomization study. J Intern Med. 2015;277:573-584
13. Klarin D, Emdin CA, Natarajan P, Conrad MF, Invent Consortium, Kathiresan S. Genetic Analysis of Venous Thromboembolism in UK Biobank Identifies the ZFPM2 Locus and Implicates Obesity as a Causal Risk Factor. Circ Cardiovasc Genet. 2017;10:e001643
14. Lindstrom S, Germain M, Crous-Bou M, Smith EN, Morange PE, van Hylckama Vlieg A, de Haan HG, Chasman D, Ridker P, Brody J, de Andrade M, Heit JA, Tang W, DeVivo I, Grodstein F, Smith NL, Tregouet D, Kabrhel C, Consortium I. Assessing the causal relationship between obesity and venous thromboembolism through a Mendelian Randomization study. Hum Genet. 2017;136:897-902
15. Larsson SC, Back M, Rees JMB, Mason AM, Burgess S. Body mass index and body composition in relation to 14 cardiovascular conditions in UK Biobank: a Mendelian randomization study. Eur Heart J. 2020;41:221-226
16. Finkelstein EA, Khavjou OA, Thompson H, Trogdon JG, Pan L, Sherry B, Dietz W. Obesity and severe obesity forecasts through 2030. Am J Prev Med. 2012;42:563-570
17. Wang Y, Beydoun MA, Liang L, Caballero B, Kumanyika SK. Will all Americans become overweight or obese? estimating the progression and cost of the US obesity epidemic. Obesity (Silver Spring). 2008;16:2323-2330
18. World Health Organization (WHO). Fact sheet: Obesity and overweight. 2018. Available at:
https://www.who.int/en/news-room/fact-sheets/detail/obesity-and-overweight. Accessed February 24, 2020
19. Arshad N, Isaksen T, Hansen JB, Braekkan SK. Time trends in incidence rates of venous thromboembolism in a large cohort recruited from the general population. Eur J Epidemiol.
2017;32:299-305
20. Heit JA, Ashrani A, Crusan DJ, McBane RD, Petterson TM, Bailey KR. Reasons for the persistent incidence of venous thromboembolism. Thromb Haemost. 2017;117:390-400
21. Münster AM, Rasmussen TB, Falstie-Jensen AM, Harboe L, Stynes G, Dybro L, Hansen ML, Brandes A, Grove EL, Johnsen SP. A changing landscape: Temporal trends in incidence and characteristics of patients hospitalized with venous thromboembolism 2006–2015.
Thrombosis Research. 2019;176:46-53
22. Glynn RJ, Rosner B. Comparison of Risk Factors for the Competing Risks of Coronary Heart Disease, Stroke, and Venous Thromboembolism. American Journal of Epidemiology.
2005;162:975-982
23. Naess IA, Christiansen SC, Romundstad P, Cannegieter SC, Rosendaal FR, Hammerstrom J.
Incidence and mortality of venous thrombosis: a population-based study. J Thromb Haemost.
2007;5:692-699
24. Heit JA. Epidemiology of venous thromboembolism. Nature Reviews Cardiology.
2015;12:464-474
25. Cohen AT, Agnelli G, Anderson FA, Arcelus JI, Bergqvist D, Brecht JG, Greer IA, Heit JA, Hutchinson JL, Kakkar AK, Mottier D, Oger E, Samama MM, Spannagl M, Europe
VTEIAGi. Venous thromboembolism (VTE) in Europe. The number of VTE events and associated morbidity and mortality. Thromb Haemost. 2007;98:756-764
26. Silverstein MD, Heit JA, Mohr DN, Petterson TM, O'Fallon WM, Melton LJ, 3rd. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Arch Intern Med. 1998;158:585-593
27. Spencer FA, Emery C, Lessard D, Anderson F, Emani S, Aragam J, Becker RC, Goldberg RJ.
The Worcester Venous Thromboembolism study: a population-based study of the clinical epidemiology of venous thromboembolism. J Gen Intern Med. 2006;21:722-727
28. Arnesen CAL, Veres K, Horváth-Puhó E, Hansen JB, Sørensen HT, Brækkan SK. Estimated lifetime risk of venous thromboembolism in men and women in a Danish nationwide cohort:
impact of competing risk of death. Eur J Epidemiol. 2021
29. Huang W, Goldberg RJ, Anderson FA, Kiefe CI, Spencer FA. Secular Trends in Occurrence of Acute Venous Thromboembolism: The Worcester VTE Study (1985-2009). The American Journal of Medicine. 2014;127:829-839.e825
30. Cushman M, Tsai AW, White RH, Heckbert SR, Rosamond WD, Enright P, Folsom AR.
Deep vein thrombosis and pulmonary embolism in two cohorts: the longitudinal investigation of thromboembolism etiology. Am J Med. 2004;117:19-25
31. Severinsen MT, Johnsen SP, Tjønneland A, Overvad K, Dethlefsen C, Kristensen SR. Body height and sex-related differences in incidence of venous thromboembolism: a Danish follow-up study. Eur J Intern Med. 2010;21:268-272
32. Roach RE, Cannegieter SC, Lijfering WM. Differential risks in men and women for first and recurrent venous thrombosis: the role of genes and environment. J Thromb Haemost.
2014;12:1593-1600
33. Roach RE, Lijfering WM, Rosendaal FR, Cannegieter SC, le Cessie S. Sex difference in risk of second but not of first venous thrombosis: paradox explained. Circulation. 2014;129:51-56 34. Yeh RW, Sidney S, Chandra M, Sorel M, Selby JV, Go AS. Population trends in the incidence
and outcomes of acute myocardial infarction. N Engl J Med. 2010;362:2155-2165
35. Vangen-Lønne AM, Wilsgaard T, Johnsen SH, Carlsson M, Mathiesen EB. Time trends in incidence and case fatality of ischemic stroke: the tromsø study 1977-2010. Stroke.
2015;46:1173-1179
36. Mannsverk J, Wilsgaard T, Mathiesen EB, Løchen ML, Rasmussen K, Thelle DS, Njølstad I, Hopstock LA, Bønaa KH. Trends in Modifiable Risk Factors Are Associated With Declining Incidence of Hospitalized and Nonhospitalized Acute Coronary Heart Disease in a Population.
Circulation. 2016;133:74-81
37. Timp JF, Braekkan SK, Versteeg HH, Cannegieter SC. Epidemiology of cancer-associated venous thrombosis. Blood. 2013;122:1712-1723
38. Rahmani J, Haghighian Roudsari A, Bawadi H, Thompson J, Khalooei Fard R, Clark C, Ryan PM, Ajami M, Rahimi Sakak F, Salehisahlabadi A, Abdulazeem HM, Jamali MR, Mirzay
39. Tagalakis V, Patenaude V, Kahn SR, Suissa S. Incidence of and mortality from venous thromboembolism in a real-world population: the Q-VTE Study Cohort. Am J Med.
2013;126:832.e813-821
40. Bikdeli B, Wang Y, Jimenez D, Parikh SA, Monreal M, Goldhaber SZ, Krumholz HM.
Pulmonary Embolism Hospitalization, Readmission, and Mortality Rates in US Older Adults, 1999-2015. Jama. 2019;322:574-576
41. Raskob GE, Angchaisuksiri P, Blanco AN, Buller H, Gallus A, Hunt BJ, Hylek EM, Kakkar A, Konstantinides SV, McCumber M, Ozaki Y, Wendelboe A, Weitz JI. Thrombosis: a major contributor to global disease burden. Arterioscler Thromb Vasc Biol. 2014;34:2363-2371 42. Grosse SD, Nelson RE, Nyarko KA, Richardson LC, Raskob GE. The economic burden of
incident venous thromboembolism in the United States: A review of estimated attributable healthcare costs. Thromb Res. 2016;137:3-10
43. van Korlaar IM, Vossen CY, Rosendaal FR, Bovill EG, Cushman M, Naud S, Kaptein AA.
The impact of venous thrombosis on quality of life. Thromb Res. 2004;114:11-18
44. Kahn SR, Shbaklo H, Lamping DL, Holcroft CA, Shrier I, Miron MJ, Roussin A, Desmarais S, Joyal F, Kassis J, Solymoss S, Desjardins L, Johri M, Ginsberg JS. Determinants of health-related quality of life during the 2 years following deep vein thrombosis. J Thromb Haemost.
2008;6:1105-1112
45. Braekkan SK, Grosse SD, Okoroh EM, Tsai J, Cannegieter SC, Naess IA, Krokstad S, Hansen JB, Skjeldestad FE. Venous thromboembolism and subsequent permanent work-related disability. J Thromb Haemost. 2016;14:1978-1987
46. Jørgensen H, Horváth-Puhó E, Laugesen K, Brækkan S, Hansen JB, Sørensen HT. Risk of a permanent work-related disability pension after incident venous thromboembolism in Denmark: A population-based cohort study. PLoS Med. 2021;18:e1003770
47. Arshad N, Bjøri E, Hindberg K, Isaksen T, Hansen JB, Brækkan SK. Recurrence and
mortality after first venous thromboembolism in a large population‐based cohort. Journal of Thrombosis and Haemostasis. 2017;15:295-303
48. Schulman S, Lindmarker P, Holmstrom M, Larfars G, Carlsson A, Nicol P, Svensson E, Ljungberg B, Viering S, Nordlander S, Leijd B, Jahed K, Hjorth M, Linder O, Beckman M.
Post-thrombotic syndrome, recurrence, and death 10 years after the first episode of venous thromboembolism treated with warfarin for 6 weeks or 6 months. Journal of Thrombosis and Haemostasis. 2006;4:734-742
49. Heit JA, Lahr BD, Ashrani AA, Petterson TM, Bailey KR. Predictors of venous
thromboembolism recurrence, adjusted for treatments and interim exposures: a population-based case-cohort study. Thromb Res. 2015;136:298-307
50. Cannegieter SC, van Hylckama Vlieg A. Venous thrombosis: understanding the paradoxes of recurrence. J Thromb Haemost. 2013;11 Suppl 1:161-169
51. Ashrani AA, Heit JA. Incidence and cost burden of post-thrombotic syndrome. J Thromb Thrombolysis. 2009;28:465-476
52. Kahn SR. The post-thrombotic syndrome. Hematology Am Soc Hematol Educ Program.
2016;2016:413-418
53. Klok FA, van der Hulle T, den Exter PL, Lankeit M, Huisman MV, Konstantinides S. The post-PE syndrome: a new concept for chronic complications of pulmonary embolism. Blood Rev. 2014;28:221-226
54. Sista AK, Klok FA. Late outcomes of pulmonary embolism: The post-PE syndrome. Thromb Res. 2018;164:157-162
55. Kahn SR, Solymoss S, Lamping DL, Abenhaim L. Long-term outcomes after deep vein thrombosis: postphlebitic syndrome and quality of life. J Gen Intern Med. 2000;15:425-429 56. Ende-Verhaar YM, Cannegieter SC, Vonk Noordegraaf A, Delcroix M, Pruszczyk P, Mairuhu
AT, Huisman MV, Klok FA. Incidence of chronic thromboembolic pulmonary hypertension after acute pulmonary embolism: a contemporary view of the published literature. Eur Respir J. 2017;49
57. Lijfering WM, Rosendaal FR, Cannegieter SC. Risk factors for venous thrombosis - current understanding from an epidemiological point of view. Br J Haematol. 2010;149:824-833
58. Heit JA. Venous thromboembolism: disease burden, outcomes and risk factors. J Thromb Haemost. 2005;3:1611-1617
59. Crous-Bou M, Harrington LB, Kabrhel C. Environmental and Genetic Risk Factors Associated with Venous Thromboembolism. Semin Thromb Hemost. 2016;42:808-820 60. Souto JC, Almasy L, Borrell M, Blanco-Vaca F, Mateo J, Soria JM, Coll I, Felices R, Stone
W, Fontcuberta J, Blangero J. Genetic susceptibility to thrombosis and its relationship to physiological risk factors: the GAIT study. Genetic Analysis of Idiopathic Thrombophilia. Am J Hum Genet. 2000;67:1452-1459
61. Heit JA, Phelps MA, Ward SA, Slusser JP, Petterson TM, De Andrade M. Familial segregation of venous thromboembolism. J Thromb Haemost. 2004;2:731-736
62. Martinelli I, De Stefano V, Mannucci PM. Inherited risk factors for venous thromboembolism.
Nat Rev Cardiol. 2014;11:140-156
63. MacArthur J, Bowler E, Cerezo M, Gil L, Hall P, Hastings E, Junkins H, McMahon A, Milano A, Morales J, Pendlington ZM, Welter D, Burdett T, Hindorff L, Flicek P, Cunningham F, Parkinson H. The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Res. 2017;45:D896-d901
64. Trégouët DA, Heath S, Saut N, Biron-Andreani C, Schved JF, Pernod G, Galan P, Drouet L, Zelenika D, Juhan-Vague I, Alessi MC, Tiret L, Lathrop M, Emmerich J, Morange PE.
Common susceptibility alleles are unlikely to contribute as strongly as the FV and ABO loci to VTE risk: results from a GWAS approach. Blood. 2009;113:5298-5303
65. Germain M, Saut N, Greliche N, et al. Genetics of venous thrombosis: insights from a new genome wide association study. PLoS One. 2011;6:e25581
66. Hinds DA, Buil A, Ziemek D, Martinez-Perez A, Malik R, Folkersen L, Germain M, Mälarstig A, Brown A, Soria JM, Dichgans M, Bing N, Franco-Cereceda A, Souto JC, Dermitzakis ET, Hamsten A, Worrall BB, Tung JY, Sabater-Lleal M. Genome-wide
association analysis of self-reported events in 6135 individuals and 252 827 controls identifies 8 loci associated with thrombosis. Hum Mol Genet. 2016;25:1867-1874
67. Lindstrom S, Wang L, Smith EN, et al. Genomic and transcriptomic association studies identify 16 novel susceptibility loci for venous thromboembolism. Blood. 2019;134:1645-1657
68. Klarin D, Busenkell E, Judy R, et al. Genome-wide association analysis of venous
thromboembolism identifies new risk loci and genetic overlap with arterial vascular disease.
Nat Genet. 2019;51:1574-1579
69. de Haan HG, Bezemer ID, Doggen CJ, Le Cessie S, Reitsma PH, Arellano AR, Tong CH, Devlin JJ, Bare LA, Rosendaal FR, Vossen CY. Multiple SNP testing improves risk prediction of first venous thrombosis. Blood. 2012;120:656-663
70. Morange PE, Tregouet DA. Lessons from genome-wide association studies in venous thrombosis. Journal of Thrombosis and Haemostasis. 2011;9:258-264
71. Germain M, Chasman DI, de Haan H, et al. Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism. Am J Hum Genet. 2015;96:532-542
72. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to
activated protein C. Nature. 1994;369:64-67
73. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88:3698-3703
74. Franchini M, Makris M. Non-O blood group: an important genetic risk factor for venous thromboembolism. Blood Transfus. 2013;11:164-165
75. Li Y, Bezemer ID, Rowland CM, Tong CH, Arellano AR, Catanese JJ, Devlin JJ, Reitsma PH, Bare LA, Rosendaal FR. Genetic variants associated with deep vein thrombosis: the F11 locus. J Thromb Haemost. 2009;7:1802-1808